Fluorovitamin D compounds and processes for their preparation

ABSTRACT

Fluorine-substituted vitamin D compounds, methods for preparation of such compounds and fluorinated intermediate compounds used in such methods are disclosed. The fluorine-substituted vitamin D compounds are characterized by vitamin D-like activity in stimulating intestinal calcium transport and bone mobilization and in promoting the calcification of rachitic bone.

This is a division of application Ser. No. 928,279, filed July 26, 1978now abandoned.

The invention described herein was made in the course of work under agrant or award from the Department of Health, Education and Welfare.

This invention relates to compounds having vitamin D-like activity. Morespecifically, this invention relates to fluoro-derivatives of vitamin D.

Vitamins D₃ and D₂ are well-known agents for the control of calcium andphosphorus homeostasis. These compounds, in the normal animal or human,stimulate intestinal-calcium transport and bone-calcium mobilization andare effective in preventing rickets. Research during the past decade hasshown, however, that vitamins D₂ and D₃ must be metabolized to theirhydroxylated forms before biological activity is expressed. Currentevidence indicates, for example, that 1,25-dihydroxyvitamin D₃, thedihydroxylated metabolite of vitamin D₃ is the compound responsible forthe biological effects mentioned earlier. Similarly,1,25-dihydroxyvitamin D₂ is the active form of vitamin D₂.

We have now found that fluorinated vitamin D compounds also possessvitamin D-like activity. These fluoro-analogs, therefore, representuseful compounds for the treatment of various diseases such asosteomalacia, osteodystrophy, and hyperparathyroidism.

This invention relates to fluorovitamin D compounds and tofluoro-5,6-trans-vitamin D compounds possessing vitamin D-like activity,and to fluorinated intermediates used for their preparation.

Specifically this invention relates to fluorovitamin D compounds ofgeneral structure I below, and 5,6-trans-fluorovitamin D compounds ofgeneral structure II below, ##STR1## where R represents a steroid sidechain of the configuration ##STR2## and where R₁ is selected from thegroup consisting of hydrogen, hydroxyl, O-lower alkyl or O-acyl, andwhere R₂, R₃, R₄ and R₅ are selected from the group consisting ofhydrogen, hydroxyl, O-lower alkyl, O-acyl, or fluoro except that atleast one of R₂, R₃, R₄, or R₅ must be fluoro, and where R₆ representshydrogen or lower alkyl.

These fluoro compounds are prepared by a process which involves thetreatment of a hydroxyvitamin D compound or hydroxyvitamin D analog witha fluorinating agent and obtaining directly the correspondingfluorovitamin D compound or fluorovitamin D analog in which fluorine islocated at the carbon originally occupied by the hydroxy function(s) ofthe starting material.

Suitable starting materials for this fluorination process includehydroxyvitamin D compounds of general structure III below, orhydroxy-5,6-trans-vitamin D compounds of general structure IV, below, orhydroxy-3,5-cyclo-vitamin D compounds of general structure V below,##STR3## where R represents a steroid side chain of the configuration##STR4## and where R₁ is selected from the group consisting of hydrogen,hydroxy, O-lower alkyl, and O-acyl, and where R₂, R₃, R₄ and R₅ areselected from hydrogen, hydroxyl, O-lower alkyl, and O-acyl, except thatat least one of R₂, R₃, R₄ or R₅ must be hydroxy, and where R₆represents hydrogen or lower alkyl, and where Z represents lower alkyl.

In this specification and in the claims the words "lower alkyl" denotesa hydrocarbon radical containing from 1 to about 5 carbon atoms whichmay be of normal or branched chain configuration (e.g. methyl, ethyl,isopropyl) and the word "acyl" denotes an aliphatic acyl groupcontaining 1 to about 5 carbon atoms (e.g. acetyl, propionyl) or anaromatic or substituted aromatic acyl group (e.g. benzoyl ornitro-benzoyl).

Fluorovitamin D compounds of general structure I are prepared fromstarting materials of general structure III, whereasfluoro-5,6-trans-vitamin D compounds of structure II are prepared fromcompounds of general structure IV, and fluorination of cyclovitamin Dstarting materials of general structure V, followed by subsequentconversion as described hereinafter leads to both fluorinated products Iand II.

The starting materials for fluorination, e.g. compounds of generalstructures III-V can be prepared by known methods. Hydroxyvitamin Dcompounds of general structure III are available by synthesis or asisolated natural products (see for example, Schnoes and DeLuca, inBioorganic Chemistry, E. E. Van Tamelen, ed., Vol., 2., Chap. 12, pp.299-335, Academic Press, N.Y., 1978). The 5,6-trans-vitamin D compoundsof general structure IV can be prepared from the corresponding 5,6-ciscompounds (general structure III) by the well-known isomerizationreaction using iodine catalyst [Verloop et al., Rec. Trav. Chim.Pays-Bas. 78, 1004 (1969)].

3,5-Cyclovitamin D compounds of general structure V can be prepared bythe procedures of Sheves and Mazur, J. Am. Chem. Soc. 97, 6249 (1975),and Paaren et al., Proc. Nat. Acad. Sci. U.S.A. 75, 2080 (1978).

Jones et al (U.S. Patent No. 4,069,321) have claimed the preparation ofvarious side chain fluorinated vitamin D₃ derivatives and side chainfluorinated dihydrotachysterol₃ analogs. The method of preparationsuggested by these investigators involves fluorination of a precursorsteroid and subsequent conversion of the fluorosteroid to the desiredfluorovitamin D compound.

It has now been found, however, that side chain and/or ringA-fluorinated vitamin D compounds or analogs of general structures I andII above can be much more conveniently prepared by direct introductionof fluorine into intact hydroxyvitamin D compounds and analogs ofgeneral structures III-V above, by means of fluorinating reagents suchas a dialkylaminosulfur trifluoride, e.g. diethylaminosulfurtrifluoride. With such reagents, one or more fluorine substituents canbe introduced into a vitamin D molecule, by direct replacement ofhydroxy function(s) in the starting material.

Middleton [J. Org. Chem. 40, 574 (1975)] has described the use ofdiethylaminosulfur trifluoride for the displacement of hydroxy functionsby fluorine in organic compounds, but his applications are limited tosimple and stable molecules, which provide no basis for judging theefficacy of the reagent for direct introduction of fluorine into vitaminD compounds without alteration of the sensitive triene chromophore.There is indeed only one previous application of diethylaminosulfurtrifluoride for the direct synthesis of 25-fluorovitamin D₃ from25-hydroxyvitamin D₃ [see Onisko, Schnoes, and DeLuca, TetrahedronLetters (no. 13) 1107 (1977)]. It is this chemical reactivity of thevitamin D chromophore that has led other investigators skilled in theart to adopt indirect and laborious routes when attempting the synthesisof fluorovitamin D compounds. For example, Jones et al. (U.S. Pat. No.4,069,321) in suggesting methods for the preparation of severalfluorovitamin D analogs, use diethylaminosulfur trifluoride only for theintroduction of fluorine into precursor steroids which are subsequentlyconverted to the fluorovitamin D analogs.

It has now been found that the aforesaid diethylaminosulfur trifluoridereagent can be used for the introduction of fluorine at variouspositions (e.g. carbon-24, 25, 26) of the side chain of a vitamin Dmolecule, as well as for the introduction of fluorine at carbon 1, andreadily permits the preparation of all the fluorovitamin D compounds ofthe Jones et al. patent (referenced above) more efficiently anddirectly.

Direct successful fluorination at carbon 1 of the vitamin D molecule isparticularly noteworthy and surprising because available informationsuggested that the vitamin triene chromophore, which is highly prone torearrangement, would undergo undesirable and irreversible alterationattendant upon fluorine introduction at carbon 1. Indeed the seeminglyanalogous reaction, namely, introduction of fluorine at carbon 3 bytreating a C-3-hydroxyvitamin D₃ compound or analog withdiethylaminosulfur trifluoride is not successful precisely because ofchromophore rearrangement. Displacement of the hydroxy function at thatposition with diethyaminosulfur trifluoride leads to an undesiredproduct in which the chromophore is altered. The facile preparation of1-fluorovitamin D compounds and vitamin D analogs is, therefore, a noveland unexpected result. It has also been observed that more than onefluorine can be introduced simultaneously simply by subjecting multiplehydroxylated (e.g. di-, trihydroxy-) vitamin D starting materials tofluorination. Since the fluorination process entails the replacement offree hydroxy function(s) it is essential that any such functions in thestarting material that are not to be replaced by fluorine by suitablyprotected, e.g. by acylation such as acetylation or benzoylation. Inparticular, a C-3-hydroxy function if present in the starting material,needs to be protected by acylation since, as already mentioned above,treatment of C-3-hydroxyvitamin D compounds or analogs withdiethylaminosulfur trifluoride leads to undesired chromophorerearrangement. Protection of hydroxy groups can be accomplished readilyby known methods and after fluorination the acyl groups can, of course,be readily removed, if desired, by hydrolysis under basic conditions.

The scope and versatility of the direct fluorination process by whichfluorovitamin D compounds of general structure I above, andfluoro-5,6-trans-vitamin D compounds of general structure II above canbe prepared from starting materials of general structures III and IV,respectively, is more specifically illustrated by the following typicalconversions.

(1) 25-hydroxyvitamin D₃ 3-O-Acyl→25-fluorovitamin D₃ 3-O-Acyl

(2) 1α,25-dihydroxyvitamin D₃ 1,3-di-O-Acyl→25-fluoro-1α-hydroxyvitaminD₃ 1,3-di-O-Acyl

(3) 1α,25-dihydroxyvitamin D₃ 3-O-Acyl→1,25-difluorovitamin D₃ 3-O-Acyl

(4) 1α-hydroxyvitamin D₃ 3-O-Acyl→1-fluorovitamin D₃ 3-O-Acyl

(5) 1α,25-dihydroxyvitamin D₃ 3,25-di-O-Acyl→1-fluoro-25-hydroxy vitaminD₃ 3,25-di-O-Acyl

(6) 24,25-dihydroxyvitamin D₃ 3,24-di-O-Acyl→25-fluoro-24-hydroxyvitaminD₃ 3,24-di-O-Acyl

(7) 1,24,25-trihydroxyvitamin D₃3,24,25-tri-O-Acyl→1-fluoro-24,25-dihydroxyvitamin D₃ 3,24,25-tri-O-Acyl

(8) 3-deoxy-1α-hydroxyvitamin D₃ →3-deoxy-1-fluorovitamin D₃

(9) 3-deoxy-1α,25-dihydroxyvitamin D₃ →3-deoxy-1,25-difluorovitamin D₃

(10) 25-hydroxyvitamin D₂ 3-O-Acyl→25-fluorovitamin D₂ 3-O-Acyl

(11) 1,25-dihydroxyvitamin D₂ 1,3-di-O-Acyl→25-fluoro-1α-hydroxyvitaminD₂ 1,3-di-O-Acyl

(12) 1,25-dihydroxyvitamin D₂ 3-O-Acyl→1,25-difluorovitamin D₂ 3-O-Acyl

(13) 25-hydroxy-5,6-trans-vitamin D₃3-O-Acyl→25-fluoro-5,6-trans-vitamin D₃ 3-O-Acyl

(14) 1α,25-dihydroxy-5,6-trans-vitamin D₃3-O-Acyl→1,25-difluoro-5,6-trans-vitamin D₃ 3-O-Acyl

(15) 25-hydroxy-5,6-trans-vitamin D₂3-O-Acyl→25-fluoro-5,6-trans-vitamin D₂ 3-O-Acyl

(16) 1,25-dihydroxy-5,6-trans-vitamin D₂3,25-di-O-Acyl→1-fluoro-25-hydroxy-5,6-trans-vitamin D₂ 3,25-di-O-Acyl

(17) 25,26-dihydroxy-5,6-trans-vitamin D₃3-O-Acyl→25,26-difluoro-5,6-trans-vitamin D₃ 3-O-Acyl

It is to be appreciated that the foregoing reactions are meant to beillustrative only. The examples are presented to show that the processprovides for the convenient introduction of fluorine into both ring Aand the side chain of the steroid molecule, and, specifically thatvitamin D compounds and analogs having fluorine substituents at any oneor more of carbons 1, 24, 25, or 26 can be readily made from thecorrespondingly hydroxylated starting materials.

A preferred reagent for fluorination is diethylaminosulfur trifluoride[Middleton, J. Org. Chem. 40, 574 (1975)]. The reaction is convenientlyconducted in a halo-carbon solvent, such as methylene chloride, carbontetrachloride or trichloromethane, at low temperature, e.g. -78° C. Forthe displacement of hydroxy groups by fluorine, short reaction times,e.g. 15-45 min, are adequate. Although the reagent also attacks ketogroups in the molecule, it does so at a much slower rate, and therefore,protection of any keto function present is not normally required underconditions where displacement of hydroxy groups is desired. It istherefore possible to fluorinate keto-vitamin D derivatives directly.For example, 1-oxoprevitamin D compounds are readily side-chainfluorinated as illustrated by reaction 18 below.

(18) 25-hydroxy-1-oxoprevitamin D₃ 3-O-Acyl→25-fluoro-1-oxo-previtaminD₃ 3-O-Acyl

1-Oxo-previtamin D compounds are readily prepared by the procedure ofPaaren et al., J. Chem. Soc. Chem. Comm. 890 (1977) and can be convertedto both 1α- and 1β-vitamin D compounds by reduction and thermalisomerization as described by Paaren et al. in the above citedreference. Thus the 25-fluoro-1-oxoprevitamin D₃ 3-O-Acyl product of thereaction above, after reduction by hydride reducing agents (e.g. LiAlH₄)yields a mixture of 1α-hydroxy-25-fluoroprevitamin D₃ and1β-hydroxy-25-fluoroprevitamin D₃, which can be separated bychromatography, and then thermally isomerized using the conditions ofPaaren et al. in the above cited reference, to the desired products,1α-hydroxy-25-fluorovitamin D₃ and 1β-hydroxy-25-fluorovitamin D₃.Fluorinated 1-oxo-previtamin D compounds, therefore, represent novel anduseful intermediates for the synthesis of desired1-hydroxy-fluorovitamin D compounds.

As mentioned previously, and as illustrated by the reaction above, anyhydroxy groups in the starting material that are not to be replaced byfluorine in this process must be protected, e.g. acylated (acetylated,benzoylated). In particular, a hydroxy function at carbon 3 (as iscommonly present in vitamin D compounds or analogs) needs to beprotected by acylation, since, as mentioned above, fluorination in thepresence of a C-3 hydroxy group can lead to undesired products.Acylation of hydroxy groups is, of course, a well-known procedure, andis normally accomplished by treating the hydroxy compound with anacylating agent (e.g. acetic anhydride, benzoyl chloride) in a suitablesolvent (e.g. pyridine). Primary and secondary hydroxy groups in vitaminD compounds or their analogs are readily acylated using such reagentsand solvents, at room temperature (or slightly elevated temperature,e.g. 50° C.), over a period of 2-6 hr. Acylation of tertiary hydroxygroups requires, of course, more vigorous conditions, e.g. elevatedtemperatures (e.g. 75°-100° C.), and appropriate reaction times, e.g.4-24 hr. It is preferable to conduct the reaction under a nitrogenatmosphere to avoid decomposition of material. Selective acylation ofspecific hydroxyl groups is also readily accomplished. Thus, by way ofexample, 25-hydroxyvitamin D₃ 3-acetate, or 1,25-dihydroxyvitamin D₃1,3-diacetate (see reactions 1 and 2, where acyl represents acetyl) canbe obtained by acetylation of 25-hydroxyvitamin D₃ and1,25-dihydroxyvitamin D₃, respectively at room temperature, since undersuch conditions the tertiary hydroxy group does not react. The acylatedstarting materials illustrated in reactions 6, 10, 11, 13, and 15 aboveare obtained similarly. Where selective acylation of chemically similarhydroxy groups is required, chromatographic separation of products maybe necessary. Thus, 1α-hydroxyvitamin D₃ 3-acetate can be prepared byconducting an acylation at room temperature and stopping the reactionbefore complete acetylation has occurred. Under such circumstances, amixture of four compounds is obtained: 1α-hydroxyvitamin D₃,1α-hydroxyvitamin D₃ 1-acetate, 1α-hydroxyvitamin D₃ 3-acetate and1α-hydroxyvitamin D₃ 1,3-diacetate, from which the desired product, e.g.1α-hydroxyvitamin D₃ 3-acetate [see reaction (4)] is obtained bychromatography. Other partially acylated products that are required asstarting materials for subsequent fluorination (e.g. as in reactions 3,12, 14) are obtained in the analogous fashion. Alternatively, partiallyacylated compounds can be obtained by first acylating all hydroxy groupsand then removing one or more of the acyl functions by hydrolysis. Thusthe 1,25-dihydroxyvitamin D₃ 3,25-di-O-acyl compound (reaction 5) can beobtained by partial hydrolysis under basic conditions of the1,25-dihydroxyvitamin D.sub. 3 1,3,25-tri-O-acyl derivative. The same3,25-di-O-acyl derivative can be prepared, if desired, by the followingroute: acylation of 25-hydroxy-6-methoxy-3,5-cyclo vitamin D₃ to producethe 25-acyl derivative, selenium dioxide oxidation of that derivative[using the mthod of Paaren et al., Proc. Nat. Acad. Sci. U.S.A. 75, 2080(1978)] to give the 1α-hydroxy analog, formylation of that derivative(using formic/acetic anhydride) to give the 1-O-formyl compound,solvolysis of that intermediate in acetic acid to yield1,25-dihydroxyvitamin D₃ 3-acetate-25-O-acyl-1-formate and finallyhydrolysis of the formate (in dilute Na₂ CO₃, for 10 minutes at roomtemperature) to produce the desired 3,25-di-O-acyl protected derivativeof 1,25-dihydroxyvitamin D₃. These examples are cited to show that manydifferent methods are known and available to produce O-protected vitaminD compounds as may be required for the subsequent fluorination reaction.

Once fluorine has been introduced into the molecule, all acyl groups, ifpresent, can be removed by basic hydrolysis, e.g. treatment of theacylated fluoro analog with 5% KOH in MeOH, at a temperature of 25°-80°C. for 1-4 hr. The resulting deacylated fluorinated products are thenconveniently further purified by chromatography.

In general, therefore, the overall process for the preparation offluorovitamin D compounds of general structures I or II from thecorresponding starting materials represented by general structures IIIor IV may involve three basic operations: (a) a preliminary protectionstep in which the C-3-hydroxy function (if present) and/or any otherhydroxy function in the starting material that is not to be replaced byfluorine is acylated; (b) the fluorination step in which fluorinereplaces any free hydroxy groups remaining after step (a), and finally,if desired (c) a deprotection step in which the protecting acyl groupsintroduced in step (a) are removed by hydrolysis under basic conditions.Of course, in those instances where the starting material contains noC-3-hydroxy function, and where all other hydroxy functions present areto be replaced by fluorine (e.g. see reactions 8 and 9 above), only asingle fluorination step is required to produce the desired product.

A novel alternative method for the preparation of fluorinated vitamin Dcompounds involves the use of cyclovitamin D compounds of generalstructure V above, as starting material. It has been found thatfluorination of 3,5-cyclovitamin D compounds of general structure Vabove is readily accomplished using the methods described previously.Fluorination of cyclovitamin D compounds of structure V represents ageneral and convenient method for the conversion of C-3-hydroxyvitamin Dcompounds to both fluorovitamin D compounds and fluoro-5,6-trans-vitaminD compounds represented respectively by general structures I and IIabove in which R₁ is selected from the group consisting of hydroxyl orO-acyl and where R, R₂, R₃, R₄, R₅ and R₆ represent substituents aspreviously defined.

These cyclovitamin D derivatives are conveniently prepared fromC-3-hydroxyvitamin D compounds by the methods published by Sheves andMazur [J. Am. Chem. Soc. 97, 6249 (1975)] and Paaren et al. [Proc. Nat.Acad. Sci. U.S.A. 75, 2080 (1978)]. Using the process of the presentinvention these compounds are readily fluorinated at carbon 1 and/or anyof the side chain positions. After fluorine introduction, thefluorocyclovitamin derivatives can be subjected to acid catalyzedsolvolysis as described by Sheves and Mazur and Paaren et al. in theabove cited references, to yield both fluorovitamin D compounds and5,6-trans-fluorovitamin D compounds which can be readily separated bychromatography. The reactions below illustrate typical conversions.##STR5##

The cyclovitamin starting materials shown in reaction 20-22 above areprepared as described by Paaren et al. in the above cited reference. Inany of the illustrated examples, step 1 represents the fluorinationreaction using diethylaminosulfur trifluoride as described earlier, andstep 2 represents acid catalyzed solvolysis using any of the conditionsof Paaren et al., in the above cited reference and step 3 representshydrolysis of acyl protecting groups (if present).

As shown by the above illustrative reactions, solvolysis yields both the5,6-cis vitamin D products and the corresponding 5,6-trans vitamin Danalogs. These fluorinated cis and trans reaction products can beconveniently separated by chromatography at this stage (as described byPaaren et al. in the above cited reference) and then can be separatelysubjected to hydrolysis (if acyl protecting groups are to be removed)using the standard conditions described earlier, e.g. 0.1 M KOH inmethanol, 60° C., 1-4 hr. Depending upon the exact solvolyzingconditions, 5,6-cis and -trans products with different C-3 substituentsare obtained. Thus, conducting the solvolysis in a medium consisting ofaqueous dioxane and a catalytic amount of p-toluene sulfonic acid yieldsfluorinated 5,6-cis and trans products bearing a C-3-hydroxysubstituent. Solvolysis of fluoro-cyclovitamin D compounds in warmacetic acid (e.g. 50°-60° C.) yields 5,6-cis and trans fluorovitamin Dcompounds bearing a C-3-acetoxy substituent, and if the solvolysis offluorocyclovitamins in conducted in dioxane/formic acid solvents, thecorresponding 3-O-formyl 5,6-cis and trans products are obtained. Ifdesired these C-3-O-acylated products can be readily converted to thecorresponding C-3-hydroxy compounds by mild base hydrolysis.

The production of both 5,6-cis and 5,6-trans-fluorovitamin D compounds(which are readily separable by chromatography) is an advantageousfeature of this novel process, and it is to be particularly noted thatthis method utilizing cyclovitamin D intermediates permits theintroduction of fluorine into either side chain positions and/or atcarbon 1 of ring A. These C-1-fluorinated 3,5-cyclovitamin D derivativesare new and highly useful intermediates.

EXAMPLE 1 25-Hydroxyvitamin D₃ 3-acetate

To 105 mg 25-hydroxyvitamin D₃ dissolved in 1.3 ml dry pyridine is added110 mg acetic anhydride. After heating at 50° C. under nitrogen for 5hr, volatiles are removed by rotary evaporation. The resulting oil isdissolved in 15 ml of ethyl acetate then washed with an equal volume of5% HCl, followed by 5% NaHCO₃, and finally saturated brine. After drying(Na₂ SO₄), the solution is applied to a silica gel thin layer plate.Development with 20% ethyl acetate in Skellysolve B and elution of themajor band with ethyl acetate gives 92.5 mg (79%) of monoacetate, i.e.25-hydroxyvitamin D₃ 3-acetate, as a colorless oil: uv (EtOH) λ_(max)265 nm (ε 15,000); ir (CCl₄) 3620 (hydroxyl), 3080 (exocyclicmethylene), 1735 and 1242 cm⁻¹ (acetate); nmr (CDCl₃) δ 6.22 (d, J=11Hz, 1H, C-6), 6.03 (d, J=11 Hz, 1H, C-7), 5.06 (d of t, J₁ =2.2 Hz, J₂=1.1 Hz, 1H, C-19), 4.94 (t of t, J₁ =4 Hz, J₂ =8.1 Hz, 1H, C-3), 4.84(d, J=2.2 Hz, 1H, C-19), 2.04 (s, 3H, acetate), 1.21 (s, 6H, C-26,27),0.93 (d, J=5.4 Hz, 3H, C-21), 0.54 (s, 3H, C-18); mass spectrum m/e(relative intensity) 442.3421 (57, M⁺, 442.3447 calcd. for C₂₉ H₄₆ O₃),382 (82), 367 (30), 253 (30), 158 (77), 118 (100), 59 (75); homogeneouson tlc (R_(f) =0.43, 20% ethyl acetate in Skellysolve B).

EXAMPLE 2

25-Fluorovitamin D₃ 3-acetate A solution of 25-hydroxyvitamin D₃3-acetate

(15 mg) in 0.5 ml of dichloromethane is added dropwise to a cooled (DryIce/2-propanol) mixture of diethylaminosulfur trifluoride (30 mg) in 0.5ml of dichloromethane. After stirring 5 min the cooling bath is removedand the contents are warmed to ambient temperature (15 min). Five ml 4%aqueuos NaHCO₃ and 10 ml dichloromethane are added. The organic phase isseparated, washed with water, dried (Na₂ SO₄), and solvents are removedby flash evaporation. This provides a yellow oil that is applied to asilica gel preparative tlc plate. After developing with 10% ethylacetate in Skellysolve B, the major product is eluted with ethylacetate. Solvent removal gives tertiary fluoride, 25-fluorovitamin D₃3-acetate (9.0 mg, 59%) as a colorless oil: uv (EtOH) λ_(max) 265 nm (ε16,00); ir (CCl₄) 3080 (exocyclic methylene), 1740 and 1240 cm⁻¹(acetate); nmr (CDCl₃) δ 6.21 (d, J=11 Hz, 1H, C-6), 6.03 (d, J=11 Hz,1H, C-7), 5.06 (m, 1H, C-19 ), 4.94 (t of t, J₁ =9 Hz, J₂ =4 Hz, 1H,C-3), 4.83 (d, J=2.2 Hz, 1H, C-19), 2.04 (s, 3H, acetate), 1.34 (d,J_(HF) =21.7 Hz, 1H, C-26,27), 0.93 (d, J=6 Hz, 3H, C=21), 0.54 (s, 3H,C-18); mass spectrum m/e (relative intensity) 444.3385 (12, M⁺, 444.3404calcd. for C₂₉ H₄₅ O₂ F) 384 (58), 369 (10), 364 (6), 253 (22), 158(44), 118 (100), 61 (22), 59 (1); homogeneous on tlc (R_(f) =0.56, 10%ethyl acetate in Skellysolve B).

EXAMPLE 3 25-Fluorovitamin D₃ 25-Fluorovitamin D₃ 3-acetate

(7.5 mg) is dissolved in 20 ml of 1.0 M KOH in methanol. After stirringat room temperature for 35 min, 5 ml water and 10 ml chloroform areadded. The phases are separated; the aqueous phase is extracted twicewith chloroform (5 ml each); the organic phases are combined then washedwith dilute acid followed by saturated brine. After drying (Na₂ SO₄),the solution is applied to a silica tlc plate. Development with 20%ethyl acetate in Skellysolve B and elution of the predominant productwith ethyl acetate provides 4.9 mg (72%) of the desired 25-fluorovitaminD₃ as a colorless oil: uv (EtOH) λ_(max) 265 nm; ir (CCl₄) 3620(hydroxyl), 3080 cm⁻¹ (exocyclic methylene); nmr (CDCl₃) δ 6.24 (d, J=11Hz, 1H, C-6), 6.03 (d, J=11 Hz, 1H, C-7), 5.05 (m, 1H, C-19), 4.82 (d,J=2.6 Hz, 1H, C-19), 3.95 (t of t, J₁ =7.1 Hz, J₂ =3.6 Hz, 1H, C-3),1.34 (d, J_(HF) --21.3 Hz, 6H, C-26,27), 0.93 (d, J=6 Hz, 3H, C-21 ),0.54 (s, 3H, C-18); mass spectrum m/e (relative intensity) 402.3284 (13,M⁺, 402.3298 calcd. for C₂₇ H₄₃ OF), 360 (4), 271 (4), 253 (5), 136(100), 118 (88), 61 (12), 59 (1); homogeneous on tlc (R_(f) =3.6 and 3.9min for the pyro and isopyro derivatives, 2 mm×6' 3% OV101, 260° ovenisothermal).

EXAMPLE 4 1α-Hydroxyvitamin D₃ 3-acetate

1α-Hydroxyvitamin D₃ (25 mg, 0.063 mmole) and acetic anhydride (50 μl)in pyridine-benzene (2 ml, 1:1) are heated at 50° C. under argon for 4hr. The reaction mixture is cooled, water and ether are added, and thephases are separated. The ether layer is washed with water, 1 N HCl (2times), dilute NaHCO₃, saturated NaCl and dried (Na₂ SO₄). The solventis evaporated and the residue is chromatographed on a silica gel plate(20×20 cm, 0.75 cm thick) developed with ethyl acetate/Skellysolve B(1:1). Four bands of R_(f) 0.20, 0.38, 0.49 and 0.64 are apparent. Theband with R_(f) 0.49 consists of product, 1α-hydroxyvitamin D₃3-acetate, 6.0 mg (22%).

The bands with R_(f) 0.64, 0.38, and 0.20 consist of 1α-hydroxyvitaminD₃ 1,3-diacetate, 1α-hydroxyvitamin D₃ 1-acetate, and starting material(7.3 mg, 29%), respectively. The undesired 1-acetate and the diacetateare combined (6.8 mg, 23-25%), hydrolyzed (1 ml 0.1 M KOH/MeOH; 1 mlether, 1.25 hr, room temperature), and pooled with starting material togive 14 mg 1α-hydroxyvitamin D₃. This process is repeated twice more togive a total of 12 mg (43%) 1α-hydroxyvitamin D₃ 3-acetate: uv (95%EtOH) λ_(max) 265, λ_(min) 228; nmr (270 MHz, CDCl₃) δ0.54 (s, 18-CH₃),0.87 (d, J=6.6 Hz, 26,27-(CH₃)₂), 0.92 (d, J=6.1 Hz, 21-CH₃), 2.03 (s, 3AcO-), 4.41 (m, 1β-H), 5.02, 5.34 (19-H's), 5.34 (3α-H), 6.02, 6.34 (ABquartet, J=11.4 Hz, 6 and 7-H's).

EXAMPLE 5 1-Fluorovitamin D₃

To 2 mg 1α-hydroxyvitamin D₃ 3β-acetate in CH₂ Cl₂ (0.4 ml) at -78° C.is added DAST (12 μl) with good stirring. The cooling bath is removedand 5 min later the reaction is quenched with 5% K₂ CO₃. Ether is addedand the phases are separated. The organic phase is washed with water,and brine, and concentrated to 0.5 ml. To the organic phase 0.1 MKOH/MeOH (1 ml) is added. After 1.5 hr at room temperature the solventis removed, ether and water are added, the phases are separated. Theether phase is washed with water and brine and filtered through Na₂ SO₄.The residue obtained after evaporation of the ether is chromatographedover a microparticulate silica gel column (5μ particles, 0.7×25 cm)eluted with 0.5% isopropanol/hexane. The desired product elutes in 78 ml(0.85 mg): uv λ_(max) 265, λ_(min) 226 nm, λ_(max) /λ_(min) 2.1; nmr(benzene-d₆) δ 0.63 (s, C-18 methyl), 0.98 (d, J=6.1 Hz, C-26,27methyls), 1.01 (d, J=6.5 Hz, C-21 methyl), 3.52 (m, w_(1/2) 24 Hz,3α-proton), 4.71 (doublet of multiplets, J=50 Hz, w_(1/2) 20 Hz), 5.235.55 (two s, 19-protons), 6.38, 6.52 (AB q, J=11 Hz, 6- and 7-protons);mass spectrum m/e (relative intensity) 402.3320 (M⁺, 0.10, calcd. forC₂₇ H₄₃ FO, 402.3298), 382 (M⁺ -HF, 0.40), 364 (M⁺ -HF-H₂ O, 0.28), 349(M⁺ -HF-H₂ O, 0.28), 349 (M⁺ -HF-H₂ O-CH₃, 0.04), 289 (M⁺ -side chain,0.05), 269 (M⁺ -side chain-HF, 0.08), 251 (M⁺ -side chain-HF-H₂ O,0.10), 135 (1.00).

EXAMPLE 6 1α,25-Dihydroxyvitamin D₃ 3-acetate

1α,25-Dihydroxyvitamin D₃ (25 mg) and acetic anhydride (50 μl) inpyridine-benzene (2 ml, 1:1) are heated at 50° C. under argon for 4 hr.The usual work-up gives a mixture of partially acetylated products(including the 1-acetate, the 3-acetate and the 1,3-diacetate) fromwhich the desired 3-acetate product is separated by preparative-layerchromatography on silica gel developed with 40% ethyl acetate inSkellysolve B. Recycling of the unreacted starting material andhydrolysis of undesired 1,3-diacetate and 1-acetate improves the overallyield.

EXAMPLE 7 1,25-Difluorovitamin D₃

1α,25-Dihydroxyvitamin D₃ 3-acetate (3 mg) in CH₂ Cl₂ at -78° C. underargon is treated with diethylaminosulfur trifluoride (20 μl). Thesolution is allowed to warm to room temperature and quenched with 5% K₂CO₃. Work-up of the reaction is done as described in Example 5.Chromatography of the recovered material over a silica gel platedeveloped with 25% ethyl acetate in Skellysolve B yields purified1,25-difluorovitamin D₃ 3-acetate. Hydrolysis (1 ml of 0.1 M KOH/MeOH, 1ml ether, room temperature, 2 hr) and rechromatography over silica gel(ethyl acetate/Skellysolve B) gives 1,25-difluorovitamin D₃.

EXAMPLE 8 1α,25-Dihydroxyvitamin D₃ 1,3-diacetate

1α,25-Dihydroxyvitamin D₃ (9.4 mg, 0.022 mmol) is heated at 50° C. for 6hr under argon with acetic anhydride (0.05 ml) and pyridine (0.3 ml) inbenzene (0.3 ml). The reaction mixture is cooled, ether and water areadded and the organic phase is separated, and washed with 1 N HCl, 5% K₂CO₃, water and brine. TLC (silica, 60% ethyl acetate/hexane) indicatedthe presence of one compound (R_(f) =0.48), the desired 1,3-diacetate of1α,25-dihydroxyvitamin D₃.

EXAMPLE 9 1α-Hydroxy-25-fluorovitamin D₃

Diethylaminosulfur trifluoride (10 μ) is added to a solution of1α,25-dihydoxyvitamin D₃ 1,3-diacetate (0.9 mg, 1.8 mmol) in carbontetrachloride (0.5 ml) at 10° C. After 10 min, the reaction is quenchedwith 5% K₂ CO₃, and chloroform is added. The organic phase is separated,washed with water and dryed (Na₂ SO₄). The residue obtained afterevaporation of the organic phase is dissolved in ether (0.5 ml) and 0.1M methanolic KOH, and is allowed to stand at room temperature for 1 hr.Ether and water are then added; the phases are separated and the organicphase is washed with water and brine. The residue is chromatographed ona high-pressure liquid chromatograph with 5% isopropanol/hexane to give86 μg of pure 1α-hydroxy-25-fluorovitamin D₃ : uv 265 nm (ε=18,200); nmr(270 MHz) 0.55 (s, 18-CH₃), 0.94 (d, J=6 Hz, 21-CH₃), 1.34 (d, J=22 Hz,26,27-(CH₃)₂), 4.23 (m, 3α-H), 4.43 (m, 1β-H), 5.33, 5.01 (2m, 19-CH₃),5.85, 6.38 (AB quartet, J=11 Hz, 6 and 7-H's); mass spectrum m/e418.3197 (M⁺, 0.07, C₂₇ H₄₄ O₂ F calcd. 418.3222), 400.3141 (M⁺ -H₂ O,0.22, calcd. 400.3143), 382.3027 (M⁺ -2×H₂ O, 0.23, calcd. 382.3031),380.3059 (M⁺ -H₂ O-HF, 0.18, calcd. 380.3069), 362.2974 (M⁺ -2×H₂ O-HF,0.18, calcd. 362.2997), 152.0842 (0.42, caldc. 152.0840), 134.0736 (1.00calcd. 134.0734).

EXAMPLE 10 24(R)-Hydroxy-25-fluorovitamin D₃

24(R),25-dihydroxyvitamin D₃ (5.0 mg) is acetylated with aceticanhydride (100 μl) and pyridine (200 μl) for 1 hr at 50° C. under argon.The reaction mixture is diluted with ether (10 ml) and washed with 1 NHCl, 5% NaHCO₃, water, and brine (5 ml each), and dried (Na₂ SO₄).Evaporation of the organic phase gives 24(R),25-dihydroxyvitamin D₃3,24-diacetate (5.4 mg): tlc (75% ethyl acetate/hexane) R_(f) 0.56(F_(f) 0.37); uv λ_(max) 265 nm, λ_(max) /λ_(min) 1.8.

The diacetate is immediately fluorinated with 50 μl diethylaminosulfurtrifluoride in 400 μl methylene chloride at -78° C. for 2 min. Thereaction is quenched by addition of 5% NaHCO₃. The mixture is extractedwith ether and treated with 0.1 M KOH/methanol for 1.5 hr at roomtemperature. The solvent is evaporated and water is added to theresidue. Extraction of the aqueous mixture with ether and evaporation ofthe organic phase yields 3.9 mg of crude products. When the mixture issubjected to high-pressure liquid chromatography (0.7×25 cm, 5μ silicagel) and eluted with 2% isopropanol/hexane, three main products areobtained. The second, eluting at 58 ml is 24(R)-hydroxy-25-fluorovitaminD₃ (831 μg): uv λ_(max) 265 nm; nmr δ 0.55 (singlet, 18-methyl), 0.94(doublet, J=5.9 Hz, 21-methyl), 1.34, 1.33 (two doublets, J=22 Hz each,26- and 27-methyl), 3.52 (multiplet, 24S-proton), 3.95 (multiplet,3α-proton), 4.82, 5.06 (multiplets, 19-protons), 6.04, 6.24 (AB quartet,J=12 Hz, 6,7-protons); mass spectrum m/e (composition, m/e calcd.)418.3264 (C₂₇ H₄₃ O₂ F, 418.3247), 398.3132 (C₂₇ H₄₂ O₂, 398.3185),385.2904 (C₂₆ H₃₈ OF, 385.2906), 271.2050 (C₁₉ H₂₇ O, 271.2061),253.1962 (C₁₉ H₂₅, 253.1956), 136.0891 (C₉ H₁₂ O, 136.0888), 118.0783(C₉ H₁₀, 118.0782).

The product eluting at 40 ml is 24(R),25-dihydroxyvitamin D₃ 24-acetate(533 μg). The compound eluting at 89 ml is24(R)-hydroxy-25-dehydrovitamin D₃ (385 μg).

EXAMPLE 11 25-Fluorovitamin D₂ -3-acetate

A solution of 2.5 mg of 25-hydroxyvitamin D₂ 3-acetate (prepared from25-hydroxyvitamin D₂ and acetic anhydride/pyridine, 50° C., 5 hr, N₂) in0.5 ml of dichloromethane is added to an excess of cold (-78° C.)diethylaminosulfur trifluoride dissolved in the same solvent. Thecooling bath is removed, and the contents are allowed to warm to roomtemperature, at which time excess reagent is destroyed with 4% NaHCO₃.The usual work-up gives an oil which is purified on a preparative tlcplate made of silica gel. Development with 10% ethyl acetate inSkellysolve B, and elution of the major band with ethyl acetate gives25-fluorovitamin D₂ 3-acetate exhibiting the expected uv, nmr, and massspectra.

EXAMPLE 12 25-Fluorovitamin D₂

25-Fluorovitamin D₂ 3-acetate (0.5 mg) is dissolved in 0.1 M methanolicKOH and kept at 25° C. for 1 hr. The reaction mixture is diluted withwater, then CHCl₃ is added and the separated organic phase isevaporated. The resulting oil is purified by silica gel preparative tlc.The plate is developed with 20% ethyl acetate in Skellysolve B and themajor band is eluted with ethyl acetate. This gives the desired product,25-fluorovitamin D₂ in good yield and purity.

EXAMPLE 13 1α-Hydroxy-25-fluorovitamin D₂

1α,25-Dihydroxyvitamin D₂ is acetylated in acetic anhydride and pyridine(4 hr, 50° C., N₂ atmosphere) to give 1α,25-dihydroxyvitamin D₂1,3-diacetate. A methylene chloride solution of this material is thenfluorinated exactly as described in Example 11. The product is isolatedas described in Example 11 and purified by thin-layer chromatography(silica gel, 10% ethyl acetate in Skellysolve B as solvent), to give1α-hydroxy-25-fluorovitamin D₂ 1,3-diacetate. Hydrolysis of this productexactly as described in Example 12 gives the desired product1α-hydroxy-25-fluorovitamin D₂.

EXAMPLE 14 25-Fluoro-1-oxoprevitamin D₃ 3-acetate

Oxidation of 1α,25-dihydroxyvitamin D₃ as described by Paaren et al. [J.Chem. Soc. Chem. Commun. 890, (1977)] yields 1-oxo-25-hydroxyprevitaminD₃. This material is acetylated by treatment with acetic anhydride inpyridine (5 hr, room temperature) to yield 1-oxo-25-hydroxyprevitamin D₃3-acetate. Fluorination of this intermediate as described in Example 11yields 25-fluoro-1-oxoprevitamin D₃ 3-acetate which is purified bypreparative thin-layer chromatography using 10% acetyl acetate andSkellysolve B as eluting solvent.

EXAMPLE 15 1,25-Dihydroxyvitamin D₃ 25-acetate

A solution of 10 mg of 1,25-dihydroxyvitamin D₃ is heated at 90° C. for16 hr under argon with 0.5 ml of acetic anhydride and 2 ml of pyridine.The usual work-up gives a mixture from which 1,25-dihydroxyvitamin D₃1,3,25-triacetate (9.5 mg) is separated by chromatography over apreparative layer of silica gel developed with 25% ethylacetate/Skellysolve B or by high-pressure liquid chromatography with asilica gel column eluted with mixtures of 0.5% 2-propanol in hexane.Selective hydrolysis (0.1 M KOH/MeOH, ether, 1.5 hr, 25° C.) of the 1and 3 acetates gives 7 mg of 1,25-dihydroxyvitamin D₃ 25-acetate, whichis purified by chromatography on silica gel thin layer plates using 50%ethyl acetate in Skellysolve B as solvent.

EXAMPLE 16 1,25-Dihydroxyvitamin D₃ 3,25-diacetate

1,25-Dihydroxyvitamin D₃ 25-acetate (7 mg) is acetylated using theconditions described in Example 4. From the product mixture1,25-dihydroxyvitamin D₃ 3,25-diacetate (4 mg) is isolated by silica gelthin layer chromatography using ethyl acetate/Skellysolve B (1:1) assolvent system.

EXAMPLE 17 1-Fluoro-25-hydroxyvitamin D₃

1,25-Dihydroxyvitamin D₃ 3,25-diacetate (2 mg) in CHCl₂ at -78° C. underargon is treated with diethylaminosulfur trifluoride (4 mg). Thereaction mixture is allowed to warm to room temperature and quenchedwith 5% K₂ CO₃. Chromatography of the recovered material over silica gelhigh-pressure liquid chromatography eluted with 1% 2-propanol in hexanegives 0.7 mg of 1-fluoro-25-hydroxyvitamin D₃ 3,25-diacetate. Hydrolysis(0.1 M KOH/MeOH, ether) and rechromatography over silica gelhigh-pressure liquid chromatography developed with 5% 2-propanol/hexaneyields 1-fluoro-25-hydroxyvitamin D₃ is pure form.

EXAMPLE 18 25-Fluoro-5,6-trans-vitamin D₃

A 5 mg sample of 25-hydroxy-5,6-trans-vitamin D₃ [Holick et al.,Biochemistry 11, 2715 (1972)] is acetylated using the conditionsdescribed in Example 1 to yield 5.2 mg of 25-hydroxyvitamin D₃3-acetate. This product is dissolved in CH₂ Cl₂ at -78° C. under argonand treated with diethylaminosulfur trifluoride (30 μl). The reaction isallowed to warm to room temperature and quenched with 5% K₂ CO₃. Afterthe usual work-up, (see Example 2) the recovered material ischromatographed by high-pressure liquid chromatography over a silica gelcolumn eluted with 0.5% of 2-propanol in hexane. Hydrolysis of theacetate (0.1 M KOH/MeOH, ether, 2 hr, 40° C.) gives25-fluoro-5,6-trans-vitamin D₃ which is purified by high-pressure liquidchromatography over a silica gel column eluted with 1% 2-propanol inhexane.

EXAMPLE 19 1-Fluoro-25-hydroxy-5,6-trans-vitamin D₃

A solution of 2 mg of 1α,25-dihydroxyvitamin D₃ 3,25-diacetate in 2 mlof ether containing a drop of pyridine is treated with 0.1 ml of asolution of iodine in Skellysolve B (0.5 mg/ml) and stirred for 15minutes. After addition of 1 ml of an aqueous solution of sodiumthiosulfate, the organic phase is separated, and solvent is evaporated.The desired product, 1α,25-dihydroxy-5,6-transvitamin D₃ 3,25-diacetateis isolated by thin layer chromatography on silica gel using 20% ethylacetate in Skellysolve B as solvent system, (yield 0.8 mg). Thismaterial is directly fluorinated using the conditions described inExample 5 (CH₂ Cl₂ solution, 10 μl diethylaminosulfur trifluoride, -78°C.) and after work-up as described in Example 5, the recovered productis hydrolyzed (0.1 M KOH, MeOH/ether, 60° C, 3 hr). Purification of thehydrolysis product by high-pressure liquid chromatography on silica gelcolumns using 3% 2-propanol in Skellysolve B as solvent yields 200 μg of1-fluoro-25-hydroxy-5,6-trans-vitamin D₃.

EXAMPLE 20 1α-Hydroxy-25-fluoro-5,6-trans-vitamin D₃

A solution of 5 mg of 1α,25-dihydroxyvitamin D₃ 1,3-diacetate in 2 ml ofether containing 2 drops of pyridine is treated with 0.2 ml of asolution of iodine in Skellysolve B (0.5 mg/ml) and stirred for 15 min.After a work-up of the reaction as described in Example 19, the desired1α,25-dihydroxy-5,6-trans-vitamin D₃ 1,3-diacetate is purified by thinlayer chromatography (silica gel plates, 10% ethyl acetate inSkellysolve B) to yield 2 mg of product. This product is fluorinatedusing the conditions of Example 9 (CH₂ Cl₂ solution, 15 μl ofdiethylaminosulfur trifluoride, -78° C., 15 min) and after work-up as inExample 5, the product is hydrolyzed (0.1 M KOH in MeOH/ether, 25° C.,1.5 hr). The hydrolysis mixture is diluted with water, and the productis extracted into ether. Purification by high-pressure liquidchromatography (silica gel column, 5% 2-propanol in hexane as solvent)gives 300 μg of 1α-hydroxy-25-fluoro-5,6-trans-vitamin D₃.

EXAMPLE 21 1α,25-Difluoro-6-methoxy-3,5-cyclovitamin D₃

A solution of 1 mg of 1,25-dihydroxy-6-methoxy-3,5-cyclovitamin D₃[prepared by the method of Paaren et al. Proc. Nat. Acad. Sci. USA, 75,2080 (1978)] in CH₂ Cl₂ at -78° C. under argon is treated withdiethylaminosulfur trifluoride (2 mg). After 10 minutes, the reactionmixture is allowed to warm to room temperature and quenched with 5% K₂CO₃. After the usual work-up, (e.g. see Example 5)1,25-difluoro-6-methoxy-3,5-cyclovitamin D₃ (250 μg) is isolated in pureform by high-pressure liquid chromatography using a silica gel columneluted with 10% tetrahydrofuran/hexane. Alternatively, the product canbe purified by thin-layer chromatography on silica gel using 20% ethylacetate in Skellysolve B as developing solvent.

EXAMPLE 22 1α,25-Dihydroxy-6-methoxy-3,5-cyclovitamin D₃ 1-acetate

1,25-Dihydroxy-6-methoxy-3,5-cyclovitamin D₃ (5 mg) prepared by themethod of Paaren et al. [Proc. Nat. Acad. Sci. USA, 75, 2080 (1978)] isacetylated with acetic anhydride (0.5 ml) and pyridine (2 ml) at 50° C.under argon for 2 hr. After the usual work-up, the 1-acetate product(5.5 mg) is isolated by high-pressure liquid chromatography over asilica gel column eluted with 2% 2-propanol/hexane.

EXAMPLE 23 25-Fluoro-1α-hydroxy-6-methoxy-3,5-cyclovitamin D₃ 1-acetate

1,25-Dihydroxy-6-methoxy-3,5-cyclovitamin D₃ 1-acetate (3 mg) in CH₂ Cl₂at -78° C. under argon is treated with diethylaminosulfur trifluoride (5mg). After several minutes, the reaction mixture is allowed to warm toroom temperature and quenched with 5% K₂ CO₃. Additional CH₂ Cl₂ isadded, the organic phase is separated and the 25-fluorinated product isisolated by thin-layer chromatography using a silica gel plate and 20%ethyl acetate in hexane as solvent, to yield 2 mg of pure product.

EXAMPLE 24 25-Fluoro-1α-hydroxy-6-methoxy-3,5-cyclovitamin D₃

A sample of 25-fluoro-cyclovitamin derivative as obtained in Example 23is dissolved in 0.1 M KOH/methanol solvent and warmed to 50° for 2 hrs.Water is then added and the hydrolyzed product is extracted into ether.Purification by thin-layer chromatography on silica gel plates developedwith 30% ethyl acetate in Skellysolve B yields pure25-fluoro-1-hydroxy-6-methoxy-3,5-cyclovitamin D₃.

EXAMPLE 25 1-Fluoro-6-methoxy-3,5-cyclovitamin D₂

A methylene chloride solution of 10 mg1α-hydroxy-6-methoxy-3,5-cyclovitamin D₂ [prepared as described byPaaren et al. Proc. Nat. Acad. Sci. USA 75, 2080 (1978)] is cooled to-78° C. and treated with 20 mg of diethylamino sulfur trifluoride. After15 minutes the solution is warmed to room temperature, aqueous NaHCO₃ (5ml) and CH₂ Cl₂ (10 ml) are added. The organic phase is separated,solvent is evaporated and the product is purified by preparativethin-layer chromatography (silica gel plates), 10% ethyl acetate inSkellysolve B as developing solvent, to yield1-fluoro-6-methoxy-3,5-cyclovitamin D₂.

EXAMPLE 26 25-Fluoro-1α-hydroxyvitamin D₃ and25-fluoro-1α-hydroxy-5,6-transvitamin D₃

A solution of 3 mg of 25-fluoro-1α-acetoxy-6-methoxy-3,5-cyclovitamin D₃[the product of Example 23] is a 3:1 mixture of dioxane and water (1.5ml) containing 0.2 mg of P-toluenesulfonic acid is warmed to 55° for 15min. Saturated NaHCO₃ (2 ml) is then added and the products areextracted into ether. The ether solvent is dried and evaporated and theresidue is chromatographed on silica gel plates to separate the 5,6-cisand 5,6-trans 25-fluorovitamin D products. Development with 25% ethylacetate in Skellysolve B gives 1 mg of pure 25-fluoro-1α-acetoxy-vitaminD₃ and 0.3 mg of 25-fluoro-1α-acetoxy-5,6-trans vitamin D₃. Hydrolysisof 25-fluoro-1α-acetoxy vitamin D₃ in 0.1 M KOH/MeOH, for 2 hrs. at 50°gives a single product, 25-fluoro-1α-hydroxyvitamin D₃. Similarhydrolysis of 25-fluoro-1α-acetoxy-5,6-transvitamin D₃ yields25-fluoro-1α-hydroxy-5,6-trans vitamin D₃ in pure form.

EXAMPLE 27 1-Fluorovitamin D₂ and 1-Fluoro-5,6-trans-vitamin D₂

A solution of 3 mg of 1-fluoro-6-methoxy-3,5-cyclovitamin D₂ [seeExample 25] in dry dioxane (2 ml) is warmed to 55° and treated with a1:1 mixture of 98% formic acid:dioxane (150 μl). After 15 min.,ice-water is added and the products are extracted with ether. Thesolvent is evaporated and the product mixture (consisting of1-fluorovitamin D₂ 3-formate and 1-fluoro-5,6-trans-vitamin D₂3-formate) is directly hydrolyzed by dissolution in dioxane:methanol(1:1) and treatment with aqueous K₂ CO₃ solution (10 mg/0.1 ml).Hydrolysis is complete after 5 min. at room temperature, and thesolution is diluted with water and the products extracted into ether.Chromatography on silica gel plates (750μ thick) using 1:3 ethylacetate:Skellysolve B as eluting solvent, separates the 5,6-cis and5,6-trans products and yields pure 1-fluorovitamin D₂ (1 mg) and1-fluoro-5,6-trans-vitamin D₂ (0.3 mg).

EXAMPLE 28 1α,24(R)25-trihydroxy-6-methoxy-3,5-cyclovitamin D₃1,24-diacetate

A pyridine solution (0.2 ml) of 10 mg of 24(R)25-dihydroxyvitamin D₃ istreated with 1.5 eg. of p-toluene sulfonyl chloride at 0° for 48 hr.Dilution with saturated NaHCO₃ solution, and extraction with etheryields crude 3-tosyl product. This product in 2 ml MeOH is treated with25 mg of NaHCO₃ and heated under N₂ at 50° for 20 hr. Dilution withwater and extraction with ether gives the cyclovitamin product.Chromatography of the product on silica gel plates (40% ethyl acetate inSkellysolve B) gives 4 mg of24(R),25-dihydroxy-6-methoxy-3,5-cyclovitamin D₃ in pure form.

This product is oxidized by treatment with SeO₂ according to theprocedures of Paaren et al. Proc. Nat. Acad. Sci. USA 75, 2080 (1978): 4mg of the cyclovitamin in CH₂ Cl₂ solution (0.5 ml) is treated with SeO₂(1 mg) and 10 μl of 70% t-butylhydroperoxide, for 30 min. at 25°. Afteraddition of NaOH solution (10%), the 1-hydroxycyclovitamin product isextracted into ether. Evaporation of solvent gives an oil which ischromatographed on silica gel plates using 50% ethyl acetate inSkellysolve B, to yield 1.5 mg of1α,24(R),25-trihydroxy-6-methoxy-3,5-cyclovitamin D₃. This product isacetylated under the usual conditions (0.2 ml of acetic anhydride in in1 ml pyridine, 50°, 2 hr). Dilution of the reaction mixture with water,extraction with ether, and evaporation of the ether yields ca. 2 mg of1α,24(R),25-trihydroxy-6-methoxy-3,5-cyclovitamin D₃ 1,24-diacetate,sufficiently pure for subsequent fluorination.

EXAMPLE 29 25-Fluoro-1,24(R)-dihydroxy-6-methoxy-3,5-cyclovitamin D₃1,24-diacetate

The diacetoxy-cyclovitamin product obtained in Example 28 is fluorinatedunder the usual conditions (CH₂ Cl₂ solvent, 3 mg of diethylaminosulfurtrifluoride, -78°, 15 min.). The reaction mixture is then allowed towarm to room temperature, quenched with 5% K₂ CO₃ solution andadditional CH₂ Cl₂ is added. The organic phase is separated and the25-fluorinated product is isolated and purified by thin-layerchromatography on silica gel plates using 25% ethyl acetate inSkellysolve B as solvent system to yield 1 mg of25-fluoro-1α,24(R)dihydroxy-6-methoxy-3,5-cyclovitamin D₃1,24-diacetate.

EXAMPLE 30 25-fluoro-1α,24(R)-dihydroxyvitamin D₃ and25-fluoro-1α,24(R)-dihydroxy-5,6-trans-vitamin D₃

A dioxane solution of the 25-fluoro cyclovitamin product as obtained inExample 29 is warmed to 55° and treated with a 1:1 mixture of dioxane:98% formic acid (200 μl). After 15 min. ice-water is added and theproducts are extracted with ether. After evaporation of solvent, theresidue is taken up in dioxane/methanol (1:1, 1 ml) and treated 0.1 mlof an aqueous K₂ CO₃ solution for 5 min. (to hydrolyze the C-3-formategroups). Extraction of the products into ether, and chromatography onsilica gel plates (using 40% ethyl acetate in Skellysolve B) yields 0.3mg of 25-fluoro-1α,24(R)-dihydroxyvitamin D₃ 1,24-diacetate and 0.1 mgof 25-fluoro-1α,24(R)-dihydroxy-5,6-trans-vitamin D₃ 1,24-diacetate. Theformer product is hydrolyzed (0.1 M KOH/MeOH, 50°,3 hr.) to25-fluoro-1α,24(R)-dihydroxyvitamin D₃. Similar hydrolysis of the5,6-trans-diacetate yields 25-fluoro-1α,24(R)-dihydroxy-5,6-trans-vitamin D₃.

Biological activity of fluorovitamin D compounds

The novel fluorovitamin D compounds prepared as described above exhibitsignificant vitamin D-like biological activity when tested in vitaminD-deficient animals. The vitamin D-like acttivities exhibited by thesefluorovitamin D compounds include the stimulation of intestinal calciumtransport, the stimulation of calcium mobilization from bone and thecalcification of bone. For the demonstration of these effects adesirable test animal is the male weanling rat maintained on a vitaminD-deficient low calcium diet, or a vitamin D-deficient, low-phosphorusdiet, as described by Suda et al. J. Nutr. 100, 1049 (1970). Withanimals maintained on a low calcium diet, intestinal calcium absorptioncan be assayed by the everted gut sac technique of Martin and DeLuca[Am. J. Physiol. 216, 1351 (1969)] and bone calcium mobilization can bedetermined by the rise of serum calcium as described for example byBlunt et al. Proc. Nat. Acad. Sci. USA 61, 1503 (1968); degree ofendochondral calcification can be assayed by the "line-test" methoddescribed in the U.S. Pharmacopeia [15th revision, p. 889, Mack Publ.Easton, Pa. (1955)] using animals maintained on the low phosphorus diet.

Using such methods the biological efficacy of the novel fluorovitamin Dcompounds of this invention is readily demonstrated. Thus, a 5 μg doseof 25-fluorovitamin D₂ administered to vitamin D-devicient,calcium-depleted animals will produce a significant stimulation ofintestinal calcium transport and elevation of serum calcium levelswithin 24 hrs after administration.

Similarly a 1 μg dose of 1α-hydroxy-25-fluorovitamin D₂ will be highlyeffective in stimulating intestinal calcium transport and bone calciummobilization (as measured by the rise of serum calcium levels), and thecompound also promotes the calcification of rachitic bone as measured bythe "line test" score.

Fluoro-5,6-trans-vitamin D compounds also exhibit pronounced vitaminD-like activity. For example 25-fluoro-5,6-trans-vitamin D₃ is effectivein stimulating intestinal calcium transport, bone calcium mobilizationand the healing of rickets in these test animals.

It has further been found that the novel 1-fluorovitamin D compounds arebiologically potent vitamin D analogs. Thus 1-fluorovitamin D₃administered intraperitoneally to rats maintained on a vitaminD-deficient low-phosphorus diet, causes significant calcification ofbone as illustrated in Table 1.

                  TABLE 1                                                         ______________________________________                                        Antirachitic Properties of 1-Fluorovitamin D.sub.3.sup.a                                     Serum phosphorus                                               Daily dose (ng).sup.b                                                                        (mg/100 ml)  Line-test score                                   ______________________________________                                        1,2-Propanediol                                                                              3.1 ± 0.3 0                                                 Vitamin D.sub.3 (20)                                                                         4.5 ± 0.3 3.6 ± 0.7                                      1-Fluorovitamin D.sub.3 (270)                                                                4.0 ± 0.2 3.2 ± 0.4                                      ______________________________________                                         .sup.a Data are expressed at mean ± SEM from 3-5 rats.                     .sup.b Test compounds given intraperitoneally in 1,2propanediol solvent       for 7 days.                                                              

Similarly, administration of 1-fluorovitamin D₃ to vitamin D-deficientanimals maintained on a low calcium diet, produces significantelevations of serum calcium levels and will effectively stimulateintestinal calcium transport, as demonstrated by the typical doseresponse data given in Table 2.

                  TABLE 2                                                         ______________________________________                                        Dose Response Data for 1-Fluorovitamin D.sub.3.sup.a                                         Bone Calcium                                                                  Mobilization                                                                  Serum calcium                                                                             Intestinal Calcium                                 Dose (ng).sup.b                                                                              (mg/100 ml) transport                                          ______________________________________                                        Ethanol (control)                                                                            4.6 ± 0.1                                                                              1.7 ± 0.2                                       Vitamin D.sub.3 (24)                                                                         5.2 ± 0.2                                                                              3.1 ± 0.3                                       1-Fluorovitamin D.sub.3 (280)                                                                5.1 ± 0.02                                                                             2.0 ± 0.2                                       1-Fluorovitamin D.sub.3 (700)                                                                5.5 ± 0.2                                                                              2.7 ± 0.2                                       1-Fluorovitamin D.sub.3 (1260)                                                               5.8 ± 0.1                                                                              3.8 ± 0.4                                       1-Fluorovitamin D.sub.3 (2450)                                                               6.2 ± 0.3                                                                              2.8 ± 0.2                                       ______________________________________                                         .sup.a Data given as mean ± SEM for 5-6 rats.                              .sup.b Compounds administered by intrajugular injections in 50 μl of       ethanol solvent.                                                         

Having thus described the invention, what is claimed is:
 1. Compoundshaving the formula ##STR6## where R₁ is selected from the groupconsisting of hydrogen, hydroxy, O-acyl and O-lower-alkyl,R₂, R₃, R₄,and R₅ are each selected from the group consisting of hydrogen, hydroxy,O-acyl, O-lower-alkyl and fluoro, except that at least one of R₂, R₃,R₄, and R₅ must be fluoro, and R₆ is hydrogen or lower alkyl.
 2. Thecompounds of claim 1 wherein R₄ is fluoro
 3. 1-fluoro-5,6-trans-vitaminD₃.
 4. 1-Fluoro-25-hydroxy-5,6-trans-vitamin D₃. 5.1,25-difluoro-5,6-trans-vitamin D₃.
 6. 25-fluoro-5,6-trans-vitamin D₃.7. 24-hydroxy-25-fluoro-5,6-trans-vitamin D₃. 8.1α-hydroxy-25-fluoro-5,6-trans-vitamin D₃. 9.1α,24(R)-dihydroxy-25-fluoro-5,6-trans-vitamin D₃.